The long term goal of this proposal is to develop a strategy of immunotherapy targeted against breast cancer stem-like cells (BCSCs). This proposal will be focused on preclinical studies to identify tumor rejection antigens expressed by murine BCSC. We will then test the translational relevance by determining whether we can identify homologous antigens in human BCSCs. We will use neu-N transgenic mice which express the oncogene rat HER-2/neu specifically in breast tissue. Neu-N mice develop spontaneous breast tumors, sometimes multiple independent tumors, recapitulating features of aggressive ER-negative, HER-2/neu positive breast tumors in human. We developed transplantable tumor lines from these spontaneous breast tumors and demonstrated that irradiated whole tumor cell vaccines could sensitize T cells in draining lymph nodes. An exciting finding is that BCSCs derived from these tumors are actually more potent vaccines for priming an immune response than the parental cell line. We have also demonstrated that neu-N mice develop IgG molecules which recognize neu-negative BCSCs following T cell adoptive immunotherapy of primary tumors, whereas pre-treatment serum is non-reactive. These data indicate that cell surface molecules on BCSCs, other than HER-2/neu, are recognized by post-treatment serum. Thus, we hypothesize that tumor-reactive CD4+ T cells provide cognate help to B cells expressing membrane Ig molecules which bind, process, and present tumor-derived antigens. The B cells acquire these otherwise sequestered antigens following destruction of tumor cells by adoptively transferred T cells. It is important to recognize that this mechanism could also lead to development of IgG responses to intracellular proteins. Therefore, to accomplish the overall objective of this proposal, two specific aims will be pursued. Specific aim 1 is to treat tumor-bearing mice with BCSC-reactive CD4+ T cells as a way to induce BCSC-specific serum. We will screen phage expression libraries derived from neu-N tumors and BCSCs with the serum thus identifying candidate BCSC antigens. We will confirm immunogenicity by introducing the relevant proteins into DCs and eliciting a response from CD4+ T cells. We will also test CD8+ T cells for reactivity to determine whether the candidate antigen has multiple intermolecular epitopes and mediates direct tumor cell recognition. Specific aim 2 is to analyze human BCSCs for expression of homologues of BCSC tumor-rejection antigens identified in mice. Immunogenicity of selected tumor antigens in humans will be assessed by introducing proteins into DCs and sensitizing T cells derived from normal donors in vitro.